Altgelt, Klaus and Hodge, Alan J. and Schmitt, Francis O. (1961) Gamma Tropocollagen: A Reversibly Denaturable Collagen Macromolecule. Proceedings of the National Academy of Sciences of the United States of America, 47 (12). pp. 1914-1924. ISSN 0027-8424 http://resolver.caltech.edu/CaltechAUTHORS:ALTpnas61
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It seemed clear that one worth-while method of investigation of the renaturable fraction would be to harvest the molecules in the form of SLS-type aggregates by centrifugation and to determine their behavior in the ultracentrifuge, after further thermal denaturation, either by running at 40° or at room temperature in the presence of KCNS. If the process of renaturation really involved a reassociation of the α and β components, then isolation of these renatured molecules via SLS formation followed by subsequent denaturation should yield a pattern in the ultracentrifuge showing both of these components in the same proportions as found in denatured solutions derived from native TC. If, on the other hand, the process of renaturation involved only a certain fraction of the macromolecules which did not dissociate into one- and two-strand components on thermal denaturation, then the process of renaturation followed by SLS harvesting should result in considerable enrichment of this minor component, and its physical characteristics should be readily apparent in the ultracentrifuge. This was indeed found to be the case. When the renaturable fraction from thermally denatured solutions of calfskin collagen was harvested as SLS by precipitation with ATP and examined in the ultracentrifuge in the presence of KCNS, it was found to consist predominantly of a single component having a sedimentation coefficient higher than both a and fi and a molecular weight equivalent to that of the native TC macromolecule, together with relatively small amounts of the α and β components. If the cycle of denaturation, renaturation, and harvesting in the form of SLS is carried out in serial fashion, the proportion of α and β components diminishes rapidly, with consequent enrichment of the higher-molecular-weight fraction, which we have termed the gamma (γ) component. As we shall see, the molecular weight and other physical properties of the γ component strongly suggest that while most of the three-strand TC macromolecules in an acid extract of collagen comprise one covalently bonded two-chain β component and one single-chain α component, a small fraction (5-10 per cent) of the macromolecules exist in a form in which the three constituent polypeptide chains are linked by covalent bonds, even in the denatured state, so that they behave as a single kinetic unit. This γ component exhibits completely reversible denaturation properties in the sense that renaturation involves a reformation of macromolecules indistinguishable in physical properties (including their charge profile as judged by electron microscopic examination of stained SLS-type aggregates) from the original native TC macromolecules. On the other hand, the α and β components, once dissociated from each other, show little if any meaningful reassociation under the same conditions of renaturation . The present paper describes some of the properties of this gamma tropocollagen, including a preliminary study of the kinetics of renaturation.
|Additional Information:||Copyright © 1961 by the National Academy of Sciences. Communicated October 31, 1961. The substance of the results given in this paper was presented at the Fifth Annual Meeting of the Biophysical Society, St. Louis, Missouri, on February 16, 1961, and at the First International Congress of Biophysics, Stockholm, Sweden, on July 31, 1961 (see abstracts of meetings). These studies were aided by research grant E-1469 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Public Health Service, U.S. Department of Health, Education, and Welfare. The technical assistance of Traudel Straub and James W. Jacques is gratefully acknowledged.|
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